Immobilization of lipase from Candida rugosa on Sepabeads(®): the effect of lipase oxidation by periodates

The objective of this paper was the investigation of a suitable Sepabeads^? support and method for immobilization of lipase from Candida rugosa. Three different supports were used, two with amino groups, (Sepabeads^? EC-EA and Sepabeads^? EC-HA), differing in spacer length (two and six carbons, respectively) and one with epoxy group (Sepabeads^? EC-EP). Lipase immobilization was carried out by two conventional methods (via epoxy groups and via glutaraldehyde), and with periodate method for modification of lipase. The results of activity assays showed that lipase retained 94.8% or 87.6% of activity after immobilization via epoxy groups or with periodate method, respectively, while glutaraldehyde method was inferior with only 12.7% of retention. The immobilization of lipase, previously modified by periodate oxidation, via amino groups has proven to be more efficient than direct immobilization of lipase via epoxy groups. In such a way immobilized enzyme exhibited higher activity at high reaction temperatures and higher thermal stability.     

Protein reutilisation in corms of Colchicum autumnale

Colchicum autumnale L. is a monocotyledonous geophyte with hysteranthous leaves, i.e. flowering and leaf growth occur in different time periods. Because after the starch, the second prominent storage compound of corm is represented by proteins, we were interested in nitrogen remobilisation during the annual life cycle of C. autumnale. In this context the content of soluble and insoluble proteins were measured in parallel with determination of some exo-and endopeptidase activities. Our results indicate that the continual proteolysis occurs in both mother and new daughter corms during the whole life-cycle of the plant. L-Ala-aminopeptidase and trypsin-like endopeptidase were the most active peptidases in both mother and daughter corms. As the protein level of mother corm did not change significantly during the development of the future above-ground part under the soil surface (the first, autumnal developmental stage), the developmental profile of nitrate reductase activity was estimated followed by evaluation of total nitrogen and amino acid contents. Significant activity of root nitrate reductase was detected in the roots only in the second, vernal stage. Our results showed that the stored proteins constituted a relevant nitrogen source partly required by the growth processes of the late autumnal stage, but mainly by the intensive growth of leaves and reproductive structures during the second, photosynthetically active stage of the life-cycle.     

Ecological Succession of a Relict Central European Peat Bog and Variability of Its Insect Biodiversity

The isolated habitat of the ervené Blato bog (South Bohemia, Czech Republic) and its relict insect fauna have been the subject of long-term monitoring. The species composition and abundance of Lepidoptera (light traps) and Coleoptera (pitfall traps) were monitored for 4 years (1994–1997) simultaneously on two sites – in the edaphic climax pine forest and in wetland successional habitats. The method of statistical evaluation by RDA and CCA ordination, representing the habitat preference of species of Coleoptera (Carabidae only) and Lepidoptera (all nocturnal phototactic taxa) between the edaphic climax forest and succession stages, was used. All categories of the peatland taxa (tyrphobiontic, tyrphophilous and tyrphoneutral species) were analysed. Ten highly stenotopic tyrphobiontic species and 23 tyrphophilous species of Lepidoptera (out of 487) were most characteristic of the bog habitat. Only two tyrphophilous carabid species (out of 20) were characteristic of the bog. The most important relict species (tyrphobionts) of Lepidoptera are most diverse and abundant within the successional habitats and in the open wet forest. The relict fauna of the closed climax pine forest is much less diverse and composed mostly of abundant tyrphophilous and tyrphoneutral forest species. Preservation or restoration of sufficiently constant hydrological conditions, which prevents formation of the closed forest, is the basic management for habitat conservation of all relict tyrphobiontic species of the ervené Blato bog and similar peat land habitat islands. The peat bog is a unified complex system of specific diverse and relict taxa. The most specific taxa are tyrphobiontic Lepidoptera, but a number of other vulnerable tyrphophilous and tyrphoneutral insects are associated with the peat bog as well.     

Littoral benthic macroinvertebrates of mountain lakes in the Tatra Mountains (Slovakia,Poland)

Littoral benthic macroinvertebrates of 45 mountain lakes in the Tatra Mountains were sampled using a semi-quantitative method in September 2000. A total of 32,852 specimens were identified to 93 taxa belonging to 14 higher taxonomic groups. Multivariate statistics (CCA, RDA) and nine biotic metrics (AQEM/STAR) were used to explain relationships between macroinvertebrate assemblages and environmental variables. Up to 57% of the ecological position of littoral macroinvertebrate assemblages were explained by variance of environmental variables divided into chemical, trophic, physical, catchment and location. Five types of Tatra lakes were recognized using CCA: A — strongly acidified lakes (small catchment, low pH, high concentration of TP, DOC, highest amount of POM in littoral); B — alpine acidified lakes (low amount of POM, low values of biotic metrics); C — alpine non-acidified lakes (high value of diversity index, predominance of Diptera); D — subalpine acidified lakes (high values of biotic metrics: number of families, proportion of crenal and rhithral taxa/total taxa); E — subalpine non-acidified lakes (high values of biotic metrics: number of families, number of genera, BMWP score, number of taxa and abundance of EPT taxa). RDA was used to design five levels of macroinvertebrate taxa acidification tolerance. The Tatra Acidification Index (TAI) was established to assess the acidification status of the lakes in the Tatra Mts.     

Effects of different detachment procedures on viability,nitroxide reduction kinetics and plasma membrane heterogeneity of V‐79 cells

Cell detachment procedures can cause severe damage to cells. Many studies require cells to be detached before measurements; therefore, research on cells that have been grown attached to the bottom of the culture dish and later detached represents a special problem with respect to the experimental results when the properties of cell membranes undergo small changes such as in spectroscopic studies of membrane permeability. We characterized the influence of three different detachment procedures: cell scraping by rubber policeman, trypsinization and a citrate buffer treatment on V‐79 cells in the plateau phase of growth (arrested in G₁). We have measured cell viability by a dye‐exclusion test; nitroxide reduction kinetics and membrane fluidity by EPR (electron paramagnetic resonance) method using the lipophilic spin‐probe MeFASL(10,3) (5‐doxylpalmitoyl‐methylester), which partitions mainly in cell membranes and the hydrophilic spin‐probe TEMPONE (4‐oxo‐2,2,6,6‐tetramethylpiperidine‐1‐oxyl). The resulting cell damage due to the detachment process was observed with SEM (scanning electron microscopy). We found out that cell viability was 91% for trypsin treatment, 85% for citrate treatment and 70% for cell scraping. Though the plasma membrane was mechanically damaged by scraping, the membrane domain structure was not significantly altered compared with other detachment methods. On the other hand, the spin‐probe reduction rate, which depends both on the transport across plasma membrane as well as on metabolic properties of cells, was the highest for trypsin method, suggesting that metabolic rate was the least influenced. Only the reduction rate of trypsin‐treated cells stayed unchanged after 4 h of stirring in suspension. These results suggest that, compared with scraping cells or using citrate buffer, the most suitable detachment method for V‐79 cells is detachment by trypsin and keeping cells in the stirred cell suspension until measurement. This method provides the highest cell viability, less visible damage on SEM micrographs and leaves the metabolic rate of cells unchanged.     

Ecological characteristics of plant species and associations along two Dinaric karstic rivers (Bosnia and Herzegovina, the Balkans)

This paper describes the plant communities of two oligotrophic karstic rivers with a slight anthropogenic influence — the Trebi?at and the Li?tica — in South Bosnia and Herzegovina, their sinecology, and the relationship between vegetation and plant species and environmental parameters. According to 87 relevés, a total of 26 plant associations, using Braun-Blanquet methods, were found in the rivers and nearby surveyed area. Only nine associations were common to both rivers. Eight associations were recorded for the territory of Bosnia and Herzegovina for the first time. Associations from both rivers differed according to following parameters measured at the sampling site: water temperature, distance from the mouth (river kilometers), river width, water depth, flow, pH and slope. There were no differences in nutrient concentrations among the associations. According to Canonical Correspondence Analysis (CCA), three environmental variables out of the 11 initially considered were retained as being related to plant distribution. The first two axes explained 56.8% and 35.2% of variance of species-environment relationship in the Trebi?at and Li?tica rivers, respectively. Water depth in the Trebi?at River, and pH and river kilometers in the Li?tica River were found to be the most influential, while nutrients and other physico-chemical parameters were not significant in either river. Water depth vector was identified as an underlying environmental factor determining distribution of Potamogeton lucens and Myriophyllum verticillatum (Potamion), and Nymphaea alba (Nymphaeion albae). According to in situ measurements of light intensity, the highest coefficients of light attenuation, radiance and reflection were found at a station with dense populations of Potamogeton lucens and Nuphar luteum (Potametum lucentis). The vegetation and floristic value of the rivers is discussed as a basis for biodiversity conservation programmes and evaluating the state of these ecosystems in the future.     

Bioinformatics analysis of bacteriophage and prophage endolysin domains

Endolysins as a class of antibacterial enzymes are expected to become a very useful tool for many purposes to control spreading of, e.g., multiresistant bacteria in different environments. Their antimicrobial properties could be broadened or altered by mutagenesis, domain swapping or gene shuffling. Therefore, the specific designing of endolysins to achieve their desired properties is challenging. This work is focused on the in silico analysis of protein domains presence in sequences of phage and prophage endolysins, followed by the study of variety of domain combinations in the individual endolysin types. The multiple sequence alignment of endolysin sequences revealed the recognition of sequence types with typical domain arrangement and conserved amino acids, divided according to the target substrate in bacterial cell walls. The five protein families of catalytic domains are specifically occurring in dependence of bacterial Gram-type. The presence, types and numbers of binding domains within endolysin sequences were also studied. The obtained results enable a more targeted design of endolysins with required antimicrobial properties.     

A Reexamination of Correlations of Amino Acids with Particular Secondary Structures

Using the data from Protein Data Bank the correlations of primary and secondary structures of proteins were analyzed. The correlation values of the amino acids and the eight secondary structure types were calculated, where the position of the amino acid and the position in sequence with the particular secondary structure differ at most 25. The diagrams describing these results indicate that correlations are significant at distances between −9 and 10. The results show that the substituents on Cβ or Cγ atoms of amino acid play major role in their preference for particular secondary structure at the same position in the sequence, while the polarity of amino acid has significant influence on α-helices and strands at some distance in the sequence. The diagrams corresponding to polar amino acids are noticeably asymmetric. The diagrams point out the exchangeability of residues in the proteins; the amino acids with similar diagrams have similar local folding requirements. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cestode systematics and phylogeny move forward

This paper represents a meeting report for the Fifth International Workshop on Cestode Systematics and Phylogeny held at the Institute of Parasitology, Academy of Sciences of the Czech Republic, České Budějovice, 18–22 July 2005. The major topics discussed included (i) the progress in cestode systematics during 2002–2005, (ii) the use of the life-cycle data in phylogenetic studies, (iii) the utilisation of new morphological and molecular characters in cestode systematics and phylogeny, and (iv) the ongoing work on the completion of the Global Cestode Database.